Learning impairment in naïve rats after intra-hippocampal cycloheximide.

<p>Intra-hippocampal cycloheximide injection (not saline) disrupted the established spatial learning (<b>A</b>: before the injection) in a parallel group of rats without ankle inflammation, as shown in two separate series of the Morris water maze test (<b>B</b>: post-injection day 1-5 and <b>C</b>: post-injection day 15-19). *P< 0.05, as compared with the intra-hippocampal vehicle group.</p

A schematic presentation of experimental designs.

<p><b>A)</b> Experiment 1 examined the effect of intra-hippocampal Aβ-induced learning impairment on the development of nociceptive behavior following ankle inflammation. <b>B</b>) Experiment 2 examined the effect of intra-hippocampal cycloheximide on the recovery of established nociceptive behavior following ankle inflammation. </p

Expression of NR1 in brain regions.

<p><b>A</b>–<b>C</b>) Immunostaining (A) and Western blot (B, C) showed an upregulation of NR1 on the ipsilateral side (to ankle inflammation) of the hippocampus in rats with the intra-hippocampal Aβ injection. Scale bar: 100 µm. <b>B</b>, <b>C</b>) In these same Aβ-injected rats, NR1 expression was downregulated in the ipsilateral thalamus but not amygdala. * <i>P</i><0.05 and ** <i>P</i><0.01, as compared with the naïve and ACSF group on the same (ipsilateral) side.</p

Expression of ChAT in the hippocampus, thalamus, and amygdala.

<p><b>A</b>) ChAT immunoreactivity was decreased in the hippocampus contralateral to ankle inflammation in Aβ group as compared with both naïve and ACSF groups with or without ankle inflammation. Veh: Vehicle. Scale bar: 100 µm. <b>B</b>, <b>C</b>) Western blot showed a substantial downregulation of ChAT expression in the contralateral hippocampus, thalamus, and amygdala of rats in Aβ group with ankle inflammation. * <i>P</i><0.05, as compared with Aβ/vehicle and Aβ/CFA groups; # <i>P</i><0.05, as compared with the ASCF/vehicle group.</p

Attenuated nociceptive behavior in rats with learning impairment.

<p><b>A</b>, <b>C</b>) The development of thermal hyperalgesia (A) and mechanical allodynia (C) was attenuated in Aβ-injected rats. <b>B</b>, <b>D</b>) No differences were detected in thermal (B) and mechanical (D) nociceptive threshold on contralateral hind paw in all groups. * <i>P</i><0.05, ** <i>P</i><0.01, as compared with baseline threshold of the same group. FWL: foot-withdrawal latency. #<0.05, # # <i>P</i><0.01, as compared with each of the remaining groups at the same time point.</p

Morris water maze test and hippocampal Aβ expression.

<p><b>A)</b> No differences were detected among three groups of rats in the hidden-platform test on day 1 and day 2. However, rats in Aβ group showed a longer escape latency than rats in ACSF group on days 3-5 and rats in naïve group on day 4-5. ** <i>P</i><0.01, as compared with ACSF group; # <i>P</i><0.01, as compared with both naïve and ACSF groups. In contrast, no differences in the escape time in visible-platform test on day 7 and 8. <b>B)</b> Immunohistochemical examination of the hippocampal CA1 area showed a substantially increased reactivity of Aβ1-40/1-42 in Aβ group, whereas Aβ immunoreactivity was barely detectable in rats of naïve and ACSF groups at the same hippocampal site. Scale bar: 100 µm. <b>C)</b> Western blot revealed a significant increase in Aβ expression in hippocampus contralateral to ankle inflammation in rats receiving Aβ injection with or without ankle inflammation. * <i>P</i><0.05, as compared with contralateral (contra) side of other groups; #<i>P<</i>0.05, as compared with ipsilateral (ipsi) side of remaining groups.</p

Disruption of persistent nociceptive behavior by cycloheximide.

<p><b>A</b>, <b>C</b>) Thermal hyperalgesia (A) and mechanical allodynia (C) were developed on the ipsilateral hind paw of the rats with ankle inflammation when tested over a 5-day period (designated as day -1 to -5). The statistical significance (ANOVA, P< 0.05; compared to the baseline) was not marked in the figure for the first five days in order to simplify the presentation. Subsequently, rats treated with cycloheximide showed a swift recovery of both thermal hyperalgesia and mechanical allodynia beginning on day 1 after cycloheximide injection. <b>B</b>, <b>D</b>) No differences in the nociceptive threshold were detected between cycloheximide and vehicle groups on the contralateral hind paw of these same rats over the entire experimental period. * <i>P</i><0.05, ** <i>P</i><0.01, as compared with baseline threshold of the same group. #<i>P</i><0.05, # # P<0.01, as compared with the cycloheximide group at the same time point. FWL: foot-withdrawal latency. White arrow: injection of CFA into an ankle; Black arrows: injection of saline (1% DMSO in saline) or cycloheximide into the hippocampus.</p

Expression of PKCγ in the brain and spinal cord.

<p><b>A</b>–<b>C</b>) Immunostaining (A) and Western blot (B, C) showed an upregulation of PKCγ in the hippocampus ipsilateral to ankle inflammation in rats with intra-hippocampal Aβ injection. Scale bar: 100 µm. <b>B</b>, <b>C</b>) In these same Aβ-injected rats, the PKCγ expression was downregulated in ipsilateral thalamus but not amygdala. In addition, the expression of PKCγ was increased within the spinal cord dorsal horn ipsilateral to ankle inflammation in both intra-hippocampal Aβ- or vehicle-injected rats. * <i>P</i><0.05, as compared with the naïve and ACSF group on the same (ipsilateral) side.</p

Image_1_Electroacupuncture attenuates surgical pain-induced delirium-like behavior in mice via remodeling gut microbiota and dendritic spine.pdf

Surgical pain is associated with delirium in patients, and acupuncture can treat pain. However, whether electroacupuncture can attenuate the surgical pain-associated delirium via the gut–brain axis remains unknown. Leveraging a mouse model of foot incision-induced surgical pain and delirium-like behavior, we found that electroacupuncture stimulation at specific acupoints (e.g., DU20+KI1) attenuated both surgical pain and delirium-like behavior in mice. Mechanistically, mice with incision-induced surgical pain and delirium-like behavior showed gut microbiota imbalance, microglia activation in the spinal cord, somatosensory cortex, and hippocampus, as well as an enhanced dendritic spine elimination in cortex revealed by two-photon imaging. The electroacupuncture regimen that alleviated surgical pain and delirium-like behavior in mice also effectively restored the gut microbiota balance, prevented the microglia activation, and reversed the dendritic spine elimination. These data demonstrated a potentially important gut–brain interactive mechanism underlying the surgical pain-induced delirium in mice. Pending further studies, these findings revealed a possible therapeutic approach in preventing and/or treating postoperative delirium by using perioperative electroacupuncture stimulation in patients.</p